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J S Richards

Publications and source records attributed to J S Richards.

At least 19 recordsLinked to original sources
PubMed1997

Expression of aromatase in the ovary: down-regulation of mRNA by the ovulatory luteinizing hormone surge.

Aromatase (CYP19) mRNA is induced by follicle-stimulating hormone (FSH) in granulosa cells of preovulatory follicles and subsequently is rapidly diminished as a consequence of the luteinizing hormone (LH) surge. Primary cultures of rat granulosa cells were used to identify some of the cellular mechanisms by which FSH increases and LH decreases steady-state levels of aromatase mRNA. Induction of aromatase mRNA by FSH was increased by cycloheximide but was blocked by alpha-amanitin and the C-kinase activators gonadotropin-releasing hormone (GnRH) and phorbol 12-myristate 13-acetate (PMA). In contrast, the decrease in steady-state levels of aromatase mRNA by LH was mimicked by A-kinase (forskolin) and C-kinase (PMA or GnRH) activators. The decrease in aromatase mRNA was associated with decreased amounts of mRNA and protein for steroidogenic factor-1 (SF-1), a nuclear orphan receptor that binds and trans-activates the aromatase promoter, and with the A-kinase subunit type II (RII beta), which is required for mediating cAMP action in these cells. The down-regulation of aromatase, SF-1, and RII beta by each kinase activator and alpha-amanitin was prevented by cycloheximide when the drug was added in combination with the activator. If, however, cycloheximide was added 2 h after PMA (or LH), the drug did not prevent the rapid loss of mRNA. When granulosa cells were transfected with an aromatase CAT transgene, CAT activity was stimulated 10- to 20-fold by FSH and forskolin but not by PMA. Taken together, these results indicate that the A-kinase but not the C-kinase pathway can trans-activate the aromatase gene in immature granulosa cells, whereas the C-kinase, as well as A-kinase pathways, mimic the LH surge to decrease aromatase mRNA in preovulatory cells. By increasing degradation of aromatase mRNA and by inhibiting transcription, the LH surge rapidly terminates the granulosa cell pattern of gene expression while reprogramming the cells to express genes associated with ovulation and luteinization.

Amanitins↗
PubMed1996

Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis.

THE D-type cyclins (D1, D2 and D3) are critical governors of the cell-cycle clock apparatus during the G1 phase of the mammalian cell cycle. These three D-type cyclins are expressed in overlapping, apparently redundant fashion in the proliferating tissues. To investigate why mammalian cells need three distinct D-type cyclins, we have generated mice bearing a disrupted cyclin D2 gene by using gene targeting in embryonic stem cells. Cyclin D2-deficient females are sterile owing to the inability of ovarian granulosa cells to proliferate normally in response to follicle-stimulating hormone (FSH), whereas mutant males display hypoplastic testes. In ovarian granulosa cells, cyclin D2 is specifically induced by FSH via a cyclic-AMP-dependent pathway, indicating that expression of the various D-type cyclins is under control of distinct intracellular signalling pathways. The hypoplasia seen in cyclin D2(-/-) ovaries and testes prompted us to examine human cancers deriving from corresponding tissues. We find that some human ovarian and testicular tumours contain high levels of cyclin D2 messenger RNA.

Animals↗
PubMed1996

An E-box region within the prostaglandin endoperoxide synthase-2 (PGS-2) promoter is required for transcription in rat ovarian granulosa cells.

The prostaglandin endoperoxide synthase-2 (PGS-2) gene encodes an isoform of prostaglandin synthase that is transiently induced by protein kinase A (luteinizing hormone/cAMP) and protein kinase C (gonadotropin-releasing hormone) agonists in granulosa cells of ovulating follicles. The promoter of the rat PGS-2 gene contains a CAAT enhancer-binding protein consensus site (CAAT box) which can confer hormone inducibility to a PGS-2.CAT reporter gene, as well as a putative E-box region. To determine if the E-box region was involved in hormone induced trans-activation of the rat PGS-2 gene, constructs with the CAAT box and E-box regions (-192 PGS-2.CAT), only the putative E-box (-110 PGS-2.CAT), or neither region (-52 PGS-2.CAT) were transiently transfected into rat granulosa cell cultures. CAT activity was induced in both the -192 and -110 PGS-2*CAT vectors by luteinizing hormone (10-fold) and gonadotropin-releasing hormone (6-fold), whereas CAT activity of the -52 PGS-2.CAT construct did not differ from the promoterless vector (pCAT-Basic). Deletion of 1 base pair from the E-box within the -110 PGS-2.CAT construct, as well as point mutations within the CAAT box, E-box, or both regions of the -192 PGS-2.CAT construct, demonstrated that the E-box is critical for basal transcription, and that regions, in addition to the CAAT box, are involved in hormone induction of the PGS-2 gene. An oligonucleotide spanning the rat PGS-2 E-box bound two specific protein complexes which were supershifted in the presence of antibody specific for the upstream stimulatory factor. Thus, in rat granulosa cells, the PGS-2 E-box region appears to interact with upstream cis-acting elements other than the CAAT box to confer hormonal regulation of the gene. The E-box region of the rat PGS-2 promoter does not contain ATF/CRE activity found in the human and mouse PGS-2 promoters, but is critical for basal transcription of the PGS-2 gene in rat granulosa cells and binds the upstream stimulatory factor (as do E-box regions of other genes regulated in the ovary).

Animals↗
PubMed1996

Effects of hormones and protein kinase inhibitors on expression of steroidogenic enzyme promoters in electroporated primary rat granulosa cells.

Previous studies have shown that inhibitors of protein tyrosine kinases, tyrphostins, can markedly attenuate the steady-state levels of mRNAs of hormone-induced genes expressed in ovarian cells. To further elucidate the mechanism of tyrphostin action, rat granulosa cells were electroporated with chimeric expression vectors containing the promoters of two key steroidogenic genes, cholesterol side chain cleavage cytochrome P450 (CYP11A; P450scc) and aromatase cytochrome P450 (CYP19; P450arom), ligated to the CAT reporter gene. The electroporation method of transfection documents that the respective promoter-reporter constructs, -379sccCAT and -534aromCAT, can confer greater than 10-fold FSH/cAMP responsiveness to the reporter genes expressed in naive granulosa cells. Furthermore, the electroporation approach allows transfection of DNA into small numbers of cells and facilitates the assay of expression in cells isolated from follicles at advanced stages of differentiation. In naive granulosa cells, the functional activities of -379sccCAT, -534aromCAT, and -169 alpha CGCAT were abolished by the A-kinase specific inhibitor, H89, supporting the notion that activation of protein kinase A is obligatory for transcriptional activation of the promoter regions within these genes. Similar inhibitory effects were also observed for tyrphostin AG18, thus implicating a tyrosine kinase in the regulation of the steroidogenic genes. As a result of eCG/hCG treatments, a gradual loss of transfection efficiency accompanied by decreasing forskolin induction of CAT expression was observed in the differentiating granulosa-lutein cells. Although the reason(s) for the apparent loss in the ability of hormones to regulate chimeric gene expression remains to be determined, cell and promoter refractoriness to hormone treatment appears to reflect a fundamental change in the mechanism of promoter activation in the differentiated cells compared to the naive granulosa cells.

Animals↗
PubMed1996

Prolactin-induced activation and binding of stat proteins to the IL-6RE of the alpha 2-macroglobulin (alpha 2M) promoter: relation to the expression of alpha 2M in the rat ovary.

Cellular signaling events by which prolactin (PRL) might regulate gene expression were analyzed in rat ovarian tissues. Whole cell extracts (WCE) were prepared from ovaries of pregnant rats (Days 4, 7, 9-11, 15, 17, and 21) and of hormonally primed (estradiol and FSH) hypophysectomized (H) immature rats before, or 15 min to 24 h after, acute administration of PRL (10 micrograms, i.v.). The DNA binding activity in the WCE was analyzed by electrophoretic mobility shift assays using the alpha 2-macroglobulin (alpha 2M) promoter interleukin (IL)-6 response element (IL-6RE) known to confer PRL and IL-6 inducibility to transgenes in target cells, including cultures of luteinized granulosa cells. Injections of PRL stimulated the appearance of a specific binding activity in ovarian extracts of H rats and in corpora lutea and interstitial extracts of pregnant rats from Days 4-9 of pregnancy. The presence of this protein/DNA complex was transient. The greater amount of binding was observed in luteinized tissue and interstitial tissue compared to follicles; and the binding activity contained specific tyrosine phosphorylated Stat (signal transduction and activators of transcription) factors identified by specific antibodies as acute phase response factor (APRF or Stat 3) and mammary gland factor (MGF, or Stat 5 [a and b]). In contrast to the transient activation and appearance of these factors in response to acute PRL treatment as administered to H rats or to pulsatile PRL release as occurs in early pregnancy, elevated levels of the same activated Stat factors were observed in WCE of CL and interstitial tissue prepared at mid-gestation (Days 10-17) when endogenous release of rat placental lactogen (rPL) is chronically elevated in serum. During this period, administration of additional exogenous PRL did not stimulate further activation (binding) of the Stat factors. During luteal regression (Day 21 of gestation) no binding was observed in the absence of PRL, and the response to PRL was markedly diminished despite the constitutive presence of Stat proteins and the Janus kinase that phosphorylates and activates these factors. Elevated binding of these factors to the IL-6RE of the alpha 2M promoter was associated with the expression of alpha 2M mRNA in luteinized granulosa cells and corpora lutea, indicating that activation of Stat factors is one mechanism by which PRL/rPL transactivates the alpha 2M gene in the tissue.

Animals↗
PubMed1996

Prolactin induction of the alpha 2-Macroglobulin gene in rat ovarian granulosa cells: stat 5 activation and binding to the interleukin-6 response element.

alpha 2-Macroglobulin (alpha 2M) is expressed at high levels in the corpus luteum of pregnant rats in response to PRL and rat placental lactogens. These studies document that PRL induction of alpha 2M mRNA occurs rapidly in granulosa cells differentiated to the preovulatory phenotype in the presence of FSH and steroid, is hormone specific [induced by PRL but not by LH or interleukin-6 (IL-6)], and involves tyrosine kinase activity. To analyze the cellular signaling events stimulated by PRL, transient transfections of granulosa cells and electrophoretic mobility shift assays were done using the IL-6 response element (IL-6RE) of the alpha 2M promoter. The IL-6RE consists of two gamma-activating like sequences (GAS) that bind the acute phase response factor (APRF/Stat 3) in rat liver and the mammary gland factor (MGF/Stat 5) from mammary tissue. By transfecting various alpha 2M promoter-luciferase reporter transgenes into the granulosa cell cultures, we show that the GAS-like sites together with the minimal -48 base pairs of the alpha 2M promoter can confer PRL inducibility to the luciferase reporter gene. These same GAS-like sequences of the alpha 2M promoter were used to analyze the DNA-binding activity of proteins in whole cell extracts prepared from differentiated granulosa cells exposed to PRL for 0.25, 0.5, 4, and 20 h. PRL rapidly stimulated the binding of a specific protein to labeled alpha 2M GAS-like oligonucleotide, and this PRL-induced binding activity was shown to contain Stat 5 but not Stat 1 or Stat 3, using specific antibodies in the electrophoretic mobility shift assays. Because both Stat 5 and Stat 3 proteins are present in the whole cell extracts of differentiated granulosa cells, PRL appears to activate detectable amounts of Stat 5 (and not Stat 3). Thus, the initial induction of the alpha 2M gene by PRL in differentiated rat granulosa cells involves, at least in part, the activation (tyrosine phosphorylation?) of Stat 5.

Animals↗
PubMed1995

Analysis of cytochrome P-450 side-chain cleavage gene promoter activation during trophoblast cell differentiation.

Trophoblast giant cell differentiation is accompanied by transcriptional activation of the cytochrome P-450 side-chain cleavage (P450scc) gene. The Rcho-1 trophoblast cell line has the capacity to differentiate along the trophoblast giant cell lineage and has been used to study trophoblast-specific P450scc gene expression. In this report, P450scc gene promoter activities in trophoblast-specific P450scc gene expression. In this report, P450scc gene promoter activities in trophoblast cells have been mapped and the involvement of known modulators of steroid hydroxylase gene expression, the cyclic AMP/protein kinase A pathway and steroidogenic factor-1 (SF-1), evaluated. Comparisons were made with Y-1 adrenal and R2C Leydig cells. The cumulative results from transient and stable transfection experiments implicate the region between -428 and -511 bp of 5'-flanking DNA in the developmental activation of the P450scc promoter during trophoblast giant cell differentiation. Differences in basal activities of the P450scc promoter constructs were also observed in Y-1 adrenal and R2C Leydig cells; however, the magnitude of the differences was modest. Activators of the protein kinase A pathway stimulated P450scc promoter activity in Y-1 cells, whereas similar treatment of Rcho-1 trophoblast cells did not stimulate but actually inhibited P450scc promoter activity. The inhibitory activity was localized between -639 and -894 bp of the P450scc promoter. SF-1 mRNA and protein were detected in adrenal and gonadal cells but not in rat placenta or Rcho-1 trophoblast cells by Northern and Western blotting, respectively. Thus, P450scc gene activation during trophoblast cell differentiation involves an 83-bp region of its 5'-flanking DNA between -428 and -511 but does not appear to involve cyclic AMP-activated pathways or SF-1. In conclusion, the mechanism of P450scc gene activation during trophoblast cell differentiation appears different from the regulation of P450scc gene activation in other steroidogenic tissues.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Ovarian cell differentiation: a cascade of multiple hormones, cellular signals, and regulated genes.

During the development of preovulatory follicles, tonic levels of FSH (and steroid) induce expression of aromatase, the LH receptor, and RII beta in a coordinate manner. Despite the similar temporal increase in steady-state levels of mRNA encoding these proteins, the cis-acting DNA elements and trans-acting factors regulating each gene are distinct (Richards, 1993). Whereas the aromatase gene has a TATA motif and a single transcriptional initiation site (Fitzpatrick and Richards, 1993), both the LH receptor (Wang et al., 1992; Tsai-Morris et al., 1993) and RII beta (Kurten et al., 1992; Luo et al., 1992) genes have promoters that are GC rich, lack TATA motifs, and initiate transcription at multiple sites. The aromatase promoter appears to be regulated, in part, by SF-1, a CRE-like region, and possibly another or overlapping region binding an Ad3BP-like factor. The RII beta promoter has a region that binds several nuclear proteins, whose identity is not yet known. Likewise, the LH receptor promoter elements have yet to be clearly defined (Figures 2, 4, and 25; Kurten et al., 1992). FSH can also induce the expression of at least three immediate-early genes that encode novel kinases or kinase-like proteins (Figure 25). One of these is called serum-inducible kinase (snk) (Simmons et al., 1992), another is serum and glucocorticoid regulated kinase (sgk) (Webster et al., 1993), and a third is called pole kinase (Clay et al., 1993). Steady-state levels of snk and sgk mRNA are induced rapidly (within a few hours) by FSH in granulosa cells prior to the appearance of transcripts for aromatase, LH receptor, and RII beta (T. Alliston and J. S. Richards, in preparation). The functional role of these kinases in the initial response of granulosa cells to tonic (not surge) levels of FSH remains to be elucidated. The cellular signaling pathways mediating the effects of the LH surge appear equally or more complex (Fig. 25). Based on data presented herein, as well as on analyses of the cloned and expressed LH receptor (Guderman et al., 1992), it is clear that low concentrations of LH stimulate adenylyl cyclase, cAMP production, and activation of protein kinase A. Higher (surge) concentrations of LH also increase IP3 and activation of protein kinase C. GnRH has been used in several studies to examine the ability of the protein kinase C pathway to mimic effects of high LH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Outcomes of post-spinal cord injury marriages.

The purpose of this study was to compare the divorce rate among persons who got married after spinal cord injury with that of the non-spinal cord-injured population of comparable age and gender and to identify factors associated with increased likelihood of divorce. The study included 622 persons enrolled in the National Spinal Cord Injury Statistical Center data set since 1973. These persons were followed between 1 and 15 years after their marriage (mean = 3.5 years). The status of each marriage was determined at the time of their most recent routine annual follow-up examination. Overall, 126 divorces occurred, whereas 74 were expected, based on 2,190 person-years of follow-up and age-sex-specific annual divorce rates for the United States population. Men and remarried persons had divorce rates 2.07 times and 1.80 times higher, respectively, than women and persons married for the first time. The divorce rate was 1.85 times higher among persons without college educations and was lower for persons with lumbosacral injuries than for persons with higher injury levels. In general, the impact of spinal cord injury appears to be almost as great on postinjury marriages as it is on preexisting marriages. However, this study yields descriptive rather than causal information. Other factors must be identified before a clinically useful model to predict persons at high risk for divorce can be developed.

Adult↗
PubMed1995

Luteinizing hormone induces prostaglandin endoperoxide synthase-2 and luteinization in vitro by A-kinase and C-kinase pathways.

The LH surge induces ovulation [prostaglandin synthase-2 (PGS-2)] and luteinization (progesterone synthesis) of preovulatory follicles by cAMP-dependent mechanisms. Peptides, such as GnRH and angiotensin-II, that activate other cellular signaling pathways have been shown to mimic some of the effects of LH. Therefore, the relative functional importance of different cellular signaling pathways in mediating the induction of PGS-2 and luteinization was analyzed using the agonists (LH, GnRH, and angiotensin-II) and selective inhibitors of A-kinase (H-89), C-kinase (calphostin-C), and calmodulin kinase-II (KN93). LH or GnRH, but not angiotensin-II, markedly induced PGS-2 protein in preovulatory follicles. H-89 and calphostin-C, but not KN93 inhibited LH induction of PGS-2, whereas calphostin-C selectively blocked induction by GnRH. In contrast, the A- and C-kinase inhibitors prevented both LH and GnRH induction of granulosa cell luteinization. Taken together, these results provide biological evidence that the response of granulosa cells to the LH surge appears to involve the activation of A- and C-kinase pathways.

Animals↗
PubMed1994

Steroidogenic factor-1 binding and transcriptional activity of the cholesterol side-chain cleavage promoter in rat granulosa cells.

The cholesterol side-chain cleavage cytochrome P450 (CYP11A; P450scc) gene is expressed in rat ovarian follicles in response to gonadotropins (FSH/LH) and cAMP. To identify functional regions within the rat P450scc promoter, 894 basepairs (bp) of 5'-flanking sequence and 5'-deletions (at -379, -101, -73, and -38 bp) were linked to the human GH reporter gene and transfected into cultured rat granulosa cells. cAMP inducibility of the rat promoter was localized to a region (between -73/-38 bp) that contains one of two AGGT/CC/TA motifs, designated SCC1 (-51/-43 bp) and SCC2 (-79/-71 bp), within the rat promoter. One of the nuclear proteins in granulosa cells that binds to SCC1 was identified as the orphan receptor, steroidogenic factor-1 (SF-1). In contrast, multiple protein-DNA complexes formed with SCC2, only one of which was clearly identified as SF-1. Nuclear extract binding was sequence specific; SCC1 bound SF-1 more strongly than did SCC2. Thus, the two AGGT/CC/TA motifs of the rat promoter appear to differ structurally and functionally. Furthermore, because the expression of SF-1 mRNA precedes hormonal/cAMP induction of P450scc mRNA and is not regulated in vitro by cAMP, the functional role of SF-1 in transcriptional regulation of the P450scc gene, including its induction by cAMP, is not entirely clear and is probably dependent on other factors and/or the modification (phosphorylation?) of SF-1.

Animals↗
PubMed1994

Tyrosine kinase inhibitor AG18 arrests follicle-stimulating hormone-induced granulosa cell differentiation: use of reverse transcriptase-polymerase chain reaction assay for multiple messenger ribonucleic acids.

A sensitive assay of multiple mRNAs by reverse transcriptase-polymerase chain reaction was adopted to study the hormonally regulated expression of steroidogenic enzymes in primary rat granulosa cells in culture. As little as 15-60 ng total RNA prepared from cultured cells were reverse transcribed in the presence of pd(T)6, and polymerase chain reaction was conducted in the presence of specific oligonucleotide pairs designed to identify cDNAs of steroidogenic enzymes. In combination with Northern blot analysis of cholesterol side-chain cleavage cytochrome P450 (P450scc) message, it is shown that a novel protein kinase inhibitor, tyrphostin AG18, arrests the FSH-induced accumulation of P450scc mRNA. This inhibition is dose dependent (IC50, 15 microM) and reversible. The addition of 80 microM AG18 to cells containing high levels of P450scc mRNA caused a rapid decline of the cytochrome message (t 1/2, 5 h), similar to the effect of 30 micrograms/ml alpha-amanitin. However, concomitant addition of the two drugs did not accelerate the mRNA degradation process, suggesting that AG18 does not affect message stabilization. Tyrphostin AG18 did not affect mRNA species that are not FSH inducible, such as the ribosomal protein L19, or the constitutively expressed low levels of steroid 5 alpha-reductase mRNA. Moreover, even the extremely high levels of P450scc mRNA in granulosa-lutein cells, being cAMP independent and terminally differentiated a few hours after LH surge, were not affected by the addition of AG18 in culture. In contrast, two additional key and FSH-inducible steroidogenic enzymes, i.e. aromatase cytochrome P450 and 3 beta-hydroxysteroid dehydrogenase-I, were inhibited by AG18 at their mRNA levels. These results suggest that an as yet undetermined tyrosine kinase pathway is involved in the cAMP-dependent signal transduction pathway of FSH action, so that the presence of AG18 does not allow FSH induction of gene expression to occur.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Identification of a cyclic adenosine 3',5'-monophosphate-response element in the rat aromatase promoter that is required for transcriptional activation in rat granulosa cells and R2C leydig cells.

Cytochrome P450 aromatase, which converts testosterone to estradiol, is transcriptionally induced by FSH and cAMP during ovarian follicular development. At least one promoter element [-82/-31 base pairs (bp)] required for stimulation of the rat gene in granulosa cells binds steroidogenic factor-1, an orphan steroid receptor. In this paper, we demonstrate that an additional region, -161/-138 bp is required for cAMP regulation. This region shares homology with promoter sequences in the bovine 21-hydroxylase and mouse 11 beta-hydroxylase genes that are also induced by cAMP, yet each binds different proteins in granulosa cell nuclear extracts. The aromatase -161/-138 bp region contains a cAMP-response element (CRE)-like sequence, TGCACGTCA. Deletion or mutation of this sequence reduces promoter activity of chimeric chloramphenicol acetyl transferase (CAT) reporter constructs that are transiently transfected into granulosa cells and R2C Leydig cells. Granulosa cell nuclear proteins and R2C cell nuclear proteins specifically bind the -161/-138 bp region and form three protein/DNA complexes. Recombinant CRE-binding protein (CREB) binds the CRE-like sequence and forms a single band, and a CREB antibody retards the migration of CREB and one granulosa cell protein-aromatase DNA binding complex. Using Western blot analysis, CREB was demonstrated in granulosa cell nuclear extracts from all stages of follicular development. Thus, aromatase is transcriptionally regulated by a hexameric sequence binding SF-1 and a CRE sequence binding CREB and other factors present in granulosa cells and in R2C Leydig cells. The presence of identical SF-1 and CRE-like sequences in the human ovarian aromatase promoter II suggests that the human promoter may also be regulated in a similar manner.

Animals↗
PubMed1993

Transcriptional regulation of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. Evidence for the role of a cis-acting C/EBP beta promoter element.

The promoter of the rat prostaglandin endoperoxide synthase 2 (PGS-2) gene has recently been shown to confer gonadotropic hormone (follicle-stimulating hormone (FSH), luteinizing hormone (LH), cAMP) inducibility when ligated to a CAT (chloramphenicol acetyltransferase) reporter gene and transfected into primary cultures of differentiated granulosa cells. To delineate cis-acting elements and trans-activating factors mediating this response, deletions of the active promoter region (-192/-53 base pairs upstream of the transcriptional start site) were tested for their ability to bind protein of granulosa cell nuclear extracts and activate reporter gene activity. Electrophoretic mobility shift assays revealed that the DNA subregion -142/-120 inhibited protein/DNA binding observed between granulosa cell nuclear extracts and the labeled PGS-2 fragment -192/-53. The subregion -142/-120 acting element C/EBP beta, 5'-TTATGCAAT-3'. Point mutations within the C/EBP beta element abolished protein/DNA binding and resulted in a 50% loss of forskolin/LH/FSH inducibility of reporter gene activity. C/EBP beta mRNA and protein were induced rapidly in granulosa cells in vivo by an ovulatory dose of human chorionic gonadotropin (hCG). Collectively, these results indicate that C/EPB beta appears to play a key role in regulating induction of the PGS-2 gene in granulosa cells prior to ovulation.

Animals↗
PubMed1993

Characterization and hormonal regulation of the promoter of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. Identification of functional and protein-binding regions.

Prostaglandin endoperoxide synthase isoform 2 (PGS-2) mRNA and protein are transiently induced by gonadotropins in granulosa cells of preovulatory follicles prior to ovulation. To better understand the hormonal regulation of the rat PGS-2 (rPGS-2) gene in these cells, genomic clones containing rPGS-2 as well as up to 6 kilobases of 5'-flanking DNA were isolated by screening a rat liver genomic library with a labeled 5'-fragment of the mouse PGS-2 cDNA. Primer extension analysis using ovarian follicular mRNA identified the presence of a single rPGS-2 transcription initiation site located 144 nucleotides upstream of the ATG translation initiation codon. To test for promoter activity within the 5'-flanking region of the rPGS-2 gene, a genomic fragment, -2698/32 (1 = cap site), as well as a series of 5'-deletion mutants, were fused upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into primary cultures of granulosa cells. Forskolin (7.5 microM), follicle-stimulating hormone (500 ng/ml) and luteinizing hormone (500 ng/ml) induced CAT activity following transfection with the -2698/32PGS.CAT, whereas gonadotropin-releasing hormone (10(-6) M) and interleukin-1 beta (30 ng/ml) had no effect. Deletion mutants delineated the region spanning from -192 to -54 of the transcription start site to be essential for both basal and forskolin-regulated expression of the reporter gene. The same DNA fragment (-192/-54) exhibited specific binding to granulosa cell nuclear extract proteins as analyzed by electrophoretic mobility shift assays. Additional specific bands were observed in extracts prepared from granulosa cells exposed to an ovulatory dose of gonadotropin. Collectively, these results provide the first structural and functional evidence that the transcriptional regulation of the rat PGS-2 gene by gonadotropins and forskolin in granulosa cells involves 5'-flanking DNA sequences, specifically a region between -192 and -54 of the transcription initiation site.

Animals↗
PubMed1993

Regulation of the rat aromatase gene in ovarian granulosa cells and R2C Leydig cells.

Aromatase cytochrome P450 is regulated in granulosa cells of ovarian follicles by the synergistic action of FSH and steroids. The effect of FSH can be mimicked by forskolin suggesting that transcription of the aromatase gene is regulated by cAMP. In contrast, aromatase is constitutively expressed in the rat R2C Leydig cells. To characterize the functional regions of the promoter in these two cell types, a fragment containing 534 bp of the aromatase promoter sequence and various deletion mutants were ligated to a reporter gene, chloramphenicol acetyl transferase and used in transient transfection assays. The results suggest that the region between -176 and -31 bp is essential both for cAMP regulation in granulosa cells and constitutive expression in R2C cells. Nuclear proteins from granulosa and R2C cells specifically bind the -176 fragment in an electrophoretic mobility shift assay. Binding was completed by an oligonucleotide (-90/-66 bp) containing a hexameric sequence, AGGTCA, which has been found in the promoters of other steroidogenic genes. These results suggest that cAMP regulation and constitutive expression of the rat aromatase promoter requires sequences between -176 and -31 bp, particularly the sequence AGGTCA at -82/.-77 and nuclear proteins binding to these sequences.

Animals↗